Bottom Line:
We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield.The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation.As a whole, we describe an optimized in vitro method for increasing OPC.

ABSTRACTNeural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC.

pone.0121774.g001: Detection of OPC markers by ICC on NS cells.A) Quantitation of NG2+ and/or PDGFRα+ cell proportions in WT mice NS generated in the presence of different growth factor combinations. Data belong to three independent experiments for each condition. Data for NG2-/PDGFRα- was analyzed with a One-way ANOVA plus Dunnett´s post test, where bFGF/EGF was set as the control. B, C) Representative images of NG2+ and PDGFRα+ cells generated from WT mice cultures in the presence of either bFGF/EGF or bFGF/PDGF-BB. D) Comparison of NG2+ or PDGFRα+ cell proportions in NS cultures generated from Act::EGFP mice in the presence of bFGF/EGF or bFGF/PDGF-BB. Data for NG2+ (dark magenta) or PDGFRα+ (light magenta) cells was analyzed separately with Student´s t test. E, F) NG2 and PDGFRα immunodetection in CNP::EGFP derived cultures. Gray bars in each graph were analyzed with Student´s t test. G) Olig2 expression in WT mice NS cultures. Asterisks are colour coded to indicate the pairs of bars compared and analyzed with Student´s t test. Cell proportions in A, and D-G are expressed as a fraction of the total cell nuclei counted for each condition. Error bars represent the SD for all bar graphs. Scale bar in C equals 100 μm for B and C. ns = not significant, * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.

Mentions:
One of the main aims of our work was to evaluate the potential of NS cells to generate oligodendrocytes (OL) under different culture conditions. We therefore analyzed NG2+ polydendrocytes and PDGFRα expressing cells by ICC to quantitate OPC proportions as a starting point. Both these markers overlapped in a high degree in this culture system. The effect of adding the standard EGF/bFGF growth factor combination during 6 days to the NS culture media was compared with the impact exerted by each factor on its own or combined with the PDGF homodimers (AA or BB). As previously observed in rat-SVZ cultures, mice NS generated NG2+ and PDGFRα+ cell populations that did not entirely overlap. After culturing NS with EGF and bFGF together, less than 20% of the cells were immunopositive for NG2 and/or PDGFRα (Fig. 1A). The highest NG2+ and/or PDGFRα+ cell proportions (more than 60%) were found in the bFGF/PDGF-BB condition, and the amount of NG2-/PDGFRα- cells was statistically different (p < 0,01) from the bFGF/EGF condition. PDGF-AA, or PDGF-BB, on its own generated non-profitable yields in terms of amount and size of NS to justify continuing with an ICC protocol, and were excluded from the analysis. Though PDGF-AA is frequently used when an OPC culture enrichment is desired, we found that OPC ratios were higher in the bFGF/PDGF-BB condition than with bFGF/PDGF-AA. The EGF/bFGF combination was used as a control (CTL) for the remaining experiments, given that it is used in most NS culture protocols published by others. The NG2+ and/or PDGFRα+ cells obtained after the CTL or bFGF/PDGF-BB treatments had a typical OPC morphology as observed by ICC (Fig. 1B, C).

pone.0121774.g001: Detection of OPC markers by ICC on NS cells.A) Quantitation of NG2+ and/or PDGFRα+ cell proportions in WT mice NS generated in the presence of different growth factor combinations. Data belong to three independent experiments for each condition. Data for NG2-/PDGFRα- was analyzed with a One-way ANOVA plus Dunnett´s post test, where bFGF/EGF was set as the control. B, C) Representative images of NG2+ and PDGFRα+ cells generated from WT mice cultures in the presence of either bFGF/EGF or bFGF/PDGF-BB. D) Comparison of NG2+ or PDGFRα+ cell proportions in NS cultures generated from Act::EGFP mice in the presence of bFGF/EGF or bFGF/PDGF-BB. Data for NG2+ (dark magenta) or PDGFRα+ (light magenta) cells was analyzed separately with Student´s t test. E, F) NG2 and PDGFRα immunodetection in CNP::EGFP derived cultures. Gray bars in each graph were analyzed with Student´s t test. G) Olig2 expression in WT mice NS cultures. Asterisks are colour coded to indicate the pairs of bars compared and analyzed with Student´s t test. Cell proportions in A, and D-G are expressed as a fraction of the total cell nuclei counted for each condition. Error bars represent the SD for all bar graphs. Scale bar in C equals 100 μm for B and C. ns = not significant, * = p < 0.05, ** = p < 0.01 and *** = p < 0.001.

Mentions:
One of the main aims of our work was to evaluate the potential of NS cells to generate oligodendrocytes (OL) under different culture conditions. We therefore analyzed NG2+ polydendrocytes and PDGFRα expressing cells by ICC to quantitate OPC proportions as a starting point. Both these markers overlapped in a high degree in this culture system. The effect of adding the standard EGF/bFGF growth factor combination during 6 days to the NS culture media was compared with the impact exerted by each factor on its own or combined with the PDGF homodimers (AA or BB). As previously observed in rat-SVZ cultures, mice NS generated NG2+ and PDGFRα+ cell populations that did not entirely overlap. After culturing NS with EGF and bFGF together, less than 20% of the cells were immunopositive for NG2 and/or PDGFRα (Fig. 1A). The highest NG2+ and/or PDGFRα+ cell proportions (more than 60%) were found in the bFGF/PDGF-BB condition, and the amount of NG2-/PDGFRα- cells was statistically different (p < 0,01) from the bFGF/EGF condition. PDGF-AA, or PDGF-BB, on its own generated non-profitable yields in terms of amount and size of NS to justify continuing with an ICC protocol, and were excluded from the analysis. Though PDGF-AA is frequently used when an OPC culture enrichment is desired, we found that OPC ratios were higher in the bFGF/PDGF-BB condition than with bFGF/PDGF-AA. The EGF/bFGF combination was used as a control (CTL) for the remaining experiments, given that it is used in most NS culture protocols published by others. The NG2+ and/or PDGFRα+ cells obtained after the CTL or bFGF/PDGF-BB treatments had a typical OPC morphology as observed by ICC (Fig. 1B, C).

Bottom Line:
We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield.The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation.As a whole, we describe an optimized in vitro method for increasing OPC.

ABSTRACTNeural Stem and Progenitor Cells (NSC/NPC) are gathering tangible recognition for their uses in cell therapy and cell replacement therapies for human disease, as well as a model system to continue research on overall neural developmental processes in vitro. The Subventricular Zone is one of the largest NSC/NPC niches in the developing mammalian Central Nervous System, and persists through to adulthood. Oligodendrocyte progenitor cell (OPC) enriched cultures are usefull tools for in vitro studies as well as for cell replacement therapies for treating demyelination diseases. We used Subventricular Zone-derived NSC/NPC primary cultures from newborn mice and compared the effects of different growth factor combinations on cell proliferation and OPC yield. The Platelet Derived Growth Factor-AA and BB homodimers had a positive and significant impact on OPC generation. Furthermore, heparin addition to the culture media contributed to further increase overall culture yields. The OPC generated by this protocol were able to mature into Myelin Basic Protein-expressing cells and to interact with neurons in an in vitro co-culture system. As a whole, we describe an optimized in vitro method for increasing OPC.